Aspects of the invention relate to methods of detection of metabolic dysfunctions using fluorescence emission from serum.
Over the past several years, fluorescence spectroscopy, a method that is several orders of magnitude more sensitive and more selective than absorption-based techniques, has been used to characterize physicochemical properties of biomolecules that exhibit fluorescence in cells, tissues and in serum. Consequently, fluorescence spectroscopy of biomolecules has been used to characterize cell metabolic pathways and to discriminate pathological conditions of cells, tissues and organs from their normal state. Previously, native fluorescence emission and excitation spectra of infected human keratinocytes, carcinoma cells, and normal human keratinocytes were measured and were shown to differ in the intracellular metabolic state of NADH. It was suggested that the observed differences were due to an increased proportion of bound mitochondrial NADH in the cancer and virus-infected cells.
In the field of diagnostic oncology, studies indicate that native fluorescence properties of tissue can be used to distinguish normal from malignant conditions in breast, cervix, colon, and bronchus samples. Measurements of emission intensity or spectral ratios of emission intensity (for example at 340 and 440 nm) under UV light excitation were shown to statistically differentiate normal from malignant tissues. The analysis of excitation spectra, monitored at the emitting wavelength (340 nm), indicated distinct differences between normal and tumor tissues.
In a series of studies, a fluorometric screening method was established for the analysis of the emission of serum to detect patients with tumors and chronic diseases. The ultraviolet fluorescence emission spectra of sera (mostly protein content) from healthy persons and of sera from cancer patients, frequently exhibited different curve shapes. Thus, to differentiate between normal sera and sera from patients suffering from neoplastic diseases, the authors analyzed emission properties in the near-ultraviolet region of the spectrum. The method developed was based on expressing the measurements obtained by the fluorescence intensity at 365 nm as a percentage of the fluorescence intensity at 337 nm. Compared with ultraviolet fluorescence emission (excitation 287 nm) of sera from healthy persons, the emission spectra of tumor sera were characterized by two regions with minor fluorescence intensities at 300 m and 340 nm and by one region of higher intensity near 325 nm. To account for the differences, the authors focused on human serum proteins, the material mainly responsible for the intrinsic fluorescence of sera in the UV spectral region. The main components that influenced the fluorescence intensity ratio were albumin and alpha-2 globulins. According to these authors, the effect may be produced by differences in the relative protein composition, which are frequently symptoms in malignancies, or tumor associated metabolites may bind to serum proteins and alter their fluorescence emission.
In addition to the complexity of observing differences in emission from different fractions in serum, fluorescence in native serum can be attributed to a variety of molecules including tryptophan (trp), tyrosine (tyr), phenylalanine (phe), NADH, pyridoxal phosphate, bilirubin, flavin-adenine dinucleotide (FAD) and others. The fluorescence associated with these molecules is defined by their concentration and distribution as well as the photo-physicochemical properties of their environment.
These previous results suggest that serum emission can be used to identify metabolic dysfunctions in humans and other animals. However, no such technique has been developed. Therefore, a study was initiated to identify a method to detect metabolic dysfunctions by fluorescence in human sera. The results reported here clearly allow one to differentiate normal from abnormal metabolic behavior in human functioning and may lead to improvement of human functioning through early detection of such disorders.
A method was identified which allows one to detect metabolic dysfunctions by fluorescence in human sera. One embodiment of this is a method for the diagnosis of a disease of metabolic dysfunction, by obtaining a sample of serum from a patient who has fasted for at least about 8 hours; irradiating the sample at an irradiation wavelength from about 300 to about 340 nm; measuring the serum emission at a wavelength from about 300 to about 600 nm; and diagnosing the presence of a metabolic dysfunction by an increased or decreased emission in comparison to an average emission at the wavelength of serum of a plurality of normal healthy volunteers.
The emission wavelength can be anywhere from about 330 to about 550, preferably, 370 to about 550, preferably from about 425 to about 500, preferably from about 470 to about 500, preferably from about 460 to about 490. Alternatively a plurality of irradiation wavelengths is used. The plurality of irradiation wavelengths can be selected from the group consisting of: 315, 325 and 340.
In one embodiment, the metabolic dysfunction is cancer and the serum emission is reduced. In a further embodiment, the patient with cancer has more than a 5% decrease in the level of serum emission. Alternatively, the patient with cancer has more than a 10% decrease in the level of serum emission. Alternatively, the patient with cancer has more than a 20% decrease in the level of serum emission. The patient with cancer may have a decrease in the serum emission from about 5% to about 60%. Alternatively, the decrease in the serum emission is from about 10% to about 50%.
In a further embodiment, the metabolic dysfunction is hypothyroidism and the serum emission is reduced. Alternatively, the metabolic dysfunction is hyperthyroidism and the serum emission is increased. Alternatively, the metabolic dysfunction is diabetes and the serum emission is reduced. In a further embodiment, the metabolic dysfunction is fatigue and the serum emission is reduced. Alternatively, the metabolic dysfunction is coronary artery disease (CAD) and the serum emission is reduced.
In a further embodiment, the sample is irradiated at both 325 nm and 340 nm. Preferably, the sample is irradiated at the wavelength which allows the best resolution for NAD(P)H.
In one embodiment, the cancer is selected from the group consisting of: breast cancer, lung cancer, colon cancer, prostate cancer and leukemia.
A further embodiment is a method for analysis of the effectiveness of a treatment, by: obtaining a first sample of serum from a patient before the treatment, wherein the patient has fasted for at least about 8 hours prior to obtaining the first sample; irradiating the first sample at an irradiation wavelength from about 300 to about 340 nm; measuring the serum emission at an emission wavelength from about 300 to about 600 nm; obtaining a second sample of serum from a patient after the treatment, wherein said patient has fasted for at least about 8 hours prior to obtaining the first sample; irradiating the second sample at an irradiation wavelength from about 300 to about 340 nm; measuring the serum emission at an emission wavelength from about 300 to about 600 nm; and diagnosing the effectiveness of the treatment by an increased or decreased emission in the second sample relative to the first sample. In one embodiment, the treatment is a treatment for cancer and the treatment is diagnosed as being effective by an increased emission.